Our long term goal is to develop a novel approach for treating established melanoma tumors by enhancing cytotoxic T cell responses towards weakly immunogenic and lowly expressed tumor antigens (TAgs) including neoantigens while simultaneously conferring T cells resistance to myeloid-derived suppressor cell (MDSC)? mediated suppression. In clinical trials, T cell-based immunotherapies have demonstrated tumor regression and increased survival rates. Yet despite these encouraging clinical results, durable antitumor responses are observed in only a subset of patients with advanced melanoma. This discrepancy stresses the need for more effective treatment strategies. T cell-based therapies are hindered in part by low T cell receptor (TCR) affinity and/or avidity and by the low expression of TAg on the tumor cell's surface. Additionally, the presence of MDSCs poses a major obstacle to generating effective and long-lived antitumor T cell responses. In our published and preliminary studies, activating MyD88 signaling in T cells, via toll like receptor engagement, augmented T cell responses and prolonged T cell survival. By fusing the CD8? (extracellular) to MyD88 (intracellular; CD8?:MyD88) molecule or by linking melanoma-reactive TCRs to MyD88 (TCR:MyD88) we have developed a novel platform that strongly activates MyD88 signaling in a TLR-independent, strictly TCR- dependent fashion. CD8?:MyD88 expression in T cells considerably amplifies responses to weak and suboptimal levels of TAg and in a TCR-dependent manner, and in preliminary studies has demonstrated the extraordinary property of differentiating MDSC's into cells that can enhance T cell responses. Importantly, the use of CD8? offers the unique feature in that it represents a `universal' approach to potentiating T cell responses, regardless of the patient's HLA type. Our central hypothesis is that T cells engineered to express a CD8?:MyD88 (or TCR:MyD88) will amplify TCR signals against a variety of weakly immunogenic or lowly expressed TAgs including neoantigens and reverse the suppressive activity of myeloid cell suppression, resulting in effective and long-lived antitumor responses. Aim 1 is to determine the molecular mechanisms by which MyD88 activation in T cells augments TCR signals. Through in vitro studies of human and mouse cells, we will define the molecular interactions through which MyD88 signaling augments TCR responses to tumor antigens of varying affinities. Aim 2 is determine the fate and antitumor efficacy of each CD8?:MyD88 and TCR:MyD88 T cells in mice with an established subcutaneous or intracranial melanoma tumor. Aim 3 is to ascertain the in vivo significance and define the mechanism(s) through which MyD88 signaling in T cells reverses myeloid cell's suppressive function. These studies are clinically significant as they have the potential to offer mechanistic insights as to how TCR responses can be amplified and, novel strategies for inhibiting and/or reversing MDSC function.